Natural occurrence of Escherichia coli-infecting bacteriophages in clinical samples

Copyright © 2019 Pacífico, Hilbert, Sofka, Dinhopl, Pap, Aspöck, Carriço and Hilbert. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.The interaction between bacteriophages, bacteria and the human host as a tripartite system has recently captured attention. The taxonomic diversity of bacteriophages, as a natural parasite of bacteria, still remains obscure in human body biomes, representing a so-called "viral dark matter." Here, we isolated and characterized coliphages from blood, urine and tracheal aspirates samples collected at a tertiary care hospital in Austria. Phages were more often isolated from blood, followed by urine and tracheal aspirates. Phylogenetic analysis and genome comparisons allowed the identification of phages belonging to the Tunavirinae subfamily, and to the Peduovirus and Tequintavirus genera. Tunavirinae phages cluster together and are found in samples from 14 patients, suggesting their prevalence across a variety of human samples. When compared with other phage genomes, the highest similarity level was at 87.69% average nucleotide identity (ANI), which suggests that these are in fact a newly isolated phage species. Tequintavirus phages share a 95.90% with phage 3_29, challenging the ANI threshold currently accepted to differentiate phage species. The isolated phages appear to be virulent, with the exception of the Peduovirus members, which are integrative and seem to reside as prophages in bacterial genomes.info:eu-repo/semantics/publishedVersio

Low-Level Tetracycline Resistance Gene tet(O)_3 in Campylobacter jejuni.

Campylobacter ( C.) spp. are the most important foodborne, bacterial, and zoonotic pathogens worldwide. Resistance monitoring of foodborne bacterial pathogens is an important tool to control antimicrobial resistance as a part of the "One Health" approach. The detection and functionality of new resistance genes are of paramount importance in applying more effective screening methods based on whole genome sequencing (WGS). Most tetracycline-resistant C. spp. isolates harbor tet(O), a gene that encodes a ribosomal protection protein. Here we describe tet(O)_3, which has been identified in two food isolates of C. jejuni and is very similar to the tet(O) gene in Streptococcus pneumoniae, having a truncated promoter sequence. This gene confers resistance to tetracycline below 1 mg/L, which is the epidemiological cut-off value. We have analyzed the entire genome of these two isolates, together with a C. jejuni isolate found to have high-level resistance to tetracycline. In contrast to the highly resistant isolate, the promoter of tet(O)_3 is highly responsive to tetracycline, as observed by reverse transcription polymerase chain reaction (RT-PCR). In addition, the two isolates possess a CRISPR repeat, fluoroquinolone resistance due to the gyrA point mutation C257T, a β-lactamase resistance gene bla OXA-184 , a multidrug efflux pump CmeABC and its repressor CmeR, but no plasmid. Low-level antibiotic resistant C. jejuni might therefore have an advantage for surviving in non-host environments

The public health risk posed by Listeria monocytogenes in frozen fruit and vegetables including herbs, blanched during processing

A multi-country outbreak ofListeria monocytogenesST6 linked to blanched frozen vegetables (bfV)took place in the EU (2015–2018). Evidence of food-borne outbreaks shows thatL. monocytogenesisthe most relevant pathogen associated with bfV. The probability of illness per serving of uncooked bfV,for the elderly (65–74 years old) population, is up to 3,600 times greater than cooked bfV and verylikely lower than any of the evaluated ready-to-eat food categories. The main factors affectingcontamination and growth ofL. monocytogenesin bfV during processing are the hygiene of the rawmaterials and process water; the hygienic conditions of the food processing environment (FPE); andthe time/Temperature (t/T) combinations used for storage and processing (e.g. blanching, cooling).Relevant factors after processing are the intrinsic characteristics of the bfV, the t/T combinations usedfor thawing and storage and subsequent cooking conditions, unless eaten uncooked. Analysis of thepossible control options suggests that application of a complete HACCP plan is either not possible orwould not further enhance food safety. Instead, specific prerequisite programmes (PRP) andoperational PRP activities should be applied such as cleaning and disinfection of the FPE, water control,t/T control and product information and consumer awareness. The occurrence of low levels ofL. monocytogenesat the end of the production process (e.g.<10 CFU/g) would be compatible with thelimit of 100 CFU/g at the moment of consumption if any labelling recommendations are strictly followed(i.e. 24 h at 5°C). Under reasonably foreseeable conditions of use (i.e. 48 h at 12°C),L. monocytogeneslevels need to be considerably lower (not detected in 25 g). Routine monitoring programmes forL. monocytogenesshould be designed following a risk-based approach and regularly revised based ontrend analysis, being FPE monitoring a key activity in the frozen vegetable industry

Microbiological safety of aged meat

The impact of dry-ageing of beef and wet-ageing of beef, pork and lamb on microbiological hazards and spoilage bacteria was examined and current practices are described. As ‘standard fresh’ and wet-aged meat use similar processes these were differentiated based on duration. In addition to a description of the different stages, data were collated on key parameters (time, temperature, pH and aw) using a literature survey and questionnaires. The microbiological hazards that may be present in all aged meats included Shiga toxin-producing Escherichia coli (STEC), Salmonella spp., Staphylococcus aureus, Listeria monocytogenes, enterotoxigenic Yersinia spp., Campylobacter spp. and Clostridium spp. Moulds, such as Aspergillus spp. and Penicillium spp., may produce mycotoxins when conditions are favourable but may be prevented by ensuring a meat surface temperature of −0.5 to 3.0°C, with a relative humidity (RH) of 75–85% and an airflow of 0.2–0.5 m/s for up to 35 days. The main meat spoilage bacteria include Pseudomonas spp., Lactobacillus spp. Enterococcus spp., Weissella spp., Brochothrix spp., Leuconostoc spp., Lactobacillus spp., Shewanella spp. and Clostridium spp. Under current practices, the ageing of meat may have an impact on the load of microbiological hazards and spoilage bacteria as compared to standard fresh meat preparation. Ageing under defined and controlled conditions can achieve the same or lower loads of microbiological hazards and spoilage bacteria than the variable log10 increases predicted during standard fresh meat preparation. An approach was used to establish the conditions of time and temperature that would achieve similar or lower levels of L. monocytogenes and Yersinia enterocolitica (pork only) and lactic acid bacteria (representing spoilage bacteria) as compared to standard fresh meat. Finally, additional control activities were identified that would further assure the microbial safety of dry-aged beef, based on recommended best practice and the outputs of the equivalence assessment.info:eu-repo/semantics/publishedVersio

Evaluation of a multi-step catalytic co-processing hydrotreatment for the production of renewable fuels using Category 3 animal fat and used cooking oils

An alternative method for the production of renewable fuels from rendered animal fats (pretreated using methods 1–5 or method 7 as described in Annex IV of Commission Regulation (EC) No 2011/142) and used cooking oils, derived from Category 3 animal by-products, was assessed. The method is based on a catalytic co-processing hydrotreatment using a middle distillate followed by a stripping step. The materials must be submitted to a pressure of at least 60 bars and a temperature of at least 270°C for at least 4.7 min. The application focuses on the demonstration of the level of reduction of spores from non-pathogenic spore-forming indicator bacterial species (Bacillus subtilis and Desulfotomaculum kuznetsovii), based on a non-systematic review of published data and additional extrapolation analyses. The EFSA BIOHAZ Panel considers that the application and supporting literature contain sufficient evidence that the proposed alternative method can achieve a reduction of at least 5 log10 in the spores of B. subtilis and a 12 log10 reduction in the spores of C. botulinum. The alternative method under evaluation is considered at least equivalent to the processing methods currently approved in the Commission Regulation (EU) No 2011/142.info:eu-repo/semantics/publishedVersio

The use of the so-called ‘superchilling’ technique for the transport of fresh fishery products

Superchilling entails lowering the fish temperature to between the initial freezing point of the fish and about 1–2°C lower. The temperature of superchilled fresh fishery products (SFFP) in boxes without ice was compared to that of products subject to the currently authorised practice in boxes with ice (CFFP) under the same conditions of on-land storage and/or transport. A heat transfer model was developed and made available as a tool to identify under which initial configurations of SFFP the fish temperature, at any time of storage/transport, is lower or equal to CFFP. A minimum degree of superchilling, corresponding to an ice fraction in the fish matrix of SFFP equal or higher than the proportion of ice added per mass of fish in CFFP, will ensure with 99–100% certainty (almost certain) that the fish temperature of SFFP and the consequent increase of relevant hazards will be lower or equal to that of CFFP. In practice, the degree of superchilling can be estimated using the fish temperature after superchilling and its initial freezing point, which are subject to uncertainties. The tool can be used as part of ‘safety-by-design’ approach, with the reliability of its outcome being dependent on the accuracy of the input data. An evaluation of methods capable of detecting whether a previously frozen fish is commercially presented as ‘superchilled’ was carried out based on, amongst others, their applicability for different fish species, ability to differentiate fresh fish from fish frozen at different temperatures, use as a stand-alone method, ease of use and classification performance. The methods that were considered ‘fit for purpose’ are Hydroxyacyl-coenzyme A dehydrogenase (HADH) test, α-glucosidase test, histology, ultraviolet–visible–near–infrared (UV-VIS/NIR) spectroscopy and hyperspectral imaging. These methods would benefit from standardisation, including the establishment of threshold values or classification algorithms to provide a practical routine test.info:eu-repo/semantics/publishedVersio

Salmonella control in poultry flocks and its public health impact

An increase in confirmed human salmonellosis cases in the EU after 2014 triggered investigation of contributory factors and control options in poultry production. Reconsideration of the five current target serovars for breeding hens showed that there is justification for retaining Salmonella Enteritidis, Salmonella Typhimurium (including monophasic variants) and Salmonella Infantis, while Salmonella Virchow and Salmonella Hadar could be replaced by Salmonella Kentucky and either Salmonella Heidelberg, Salmonella Thompson or a variable serovar in national prevalence targets. However, a target that incorporates all serovars is expected to be more effective as the most relevant serovars in breeding flocks vary between Member State (MS) and over time. Achievement of a 1% target for the current target serovars in laying hen flocks is estimated to be reduced by 254,400 CrI95[98,540; 602,700] compared to the situation in 2016. This translates to a reduction of 53.4% CrI95[39.1; 65.7] considering the layer-associated human salmonellosis true cases and 6.2% considering the overall human salmonellosis true cases in the 23 MSs included in attribution modelling. A review of risk factors for Salmonella in laying hens revealed that overall evidence points to a lower occurrence in non-cage compared to cage systems. A conclusion on the effect of outdoor access or impact of the shift from conventional to enriched cages could not be reached. A similar review for broiler chickens concluded that the evidence that outdoor access affects the occurrence of Salmonella is inconclusive. There is conclusive evidence that an increased stocking density, larger farms and stress result in increased occurrence, persistence and spread of Salmonella in laying hen flocks. Based on scientific evidence, an impact of Salmonella control programmes, apart from general hygiene procedures, on the prevalence of Campylobacter in broiler flocks at the holding and on broiler meat at the end of the slaughter process is not expected

Update and review of control options for Campylobacter in broilers at primary production

The 2011 EFSA opinion on Campylobacter was updated using more recent scientific data. The relative risk reduction in EU human campylobacteriosis attributable to broiler meat was estimated for on‐farm control options using Population Attributable Fractions (PAF) for interventions that reduce Campylobacter flock prevalence, updating the modelling approach for interventions that reduce caecal concentrations and reviewing scientific literature. According to the PAF analyses calculated for six control options, the mean relative risk reductions that could be achieved by adoption of each of these six control options individually are estimated to be substantial but the width of the confidence intervals of all control options indicates a high degree of uncertainty in the specific risk reduction potentials. The updated model resulted in lower estimates of impact than the model used in the previous opinion. A 3‐log10 reduction in broiler caecal concentrations was estimated to reduce the relative EU risk of human campylobacteriosis attributable to broiler meat by 58% compared to an estimate larger than 90% in the previous opinion. Expert Knowledge Elicitation was used to rank control options, for weighting and integrating different evidence streams and assess uncertainties. Medians of the relative risk reductions of selected control options had largely overlapping probability intervals, so the rank order was uncertain: vaccination 27% (90% probability interval (PI) 4–74%); feed and water additives 24% (90% PI 4–60%); discontinued thinning 18% (90% PI 5–65%); employing few and well‐trained staff 16% (90% PI 5–45%); avoiding drinkers that allow standing water 15% (90% PI 4–53%); addition of disinfectants to drinking water 14% (90% PI 3–36%); hygienic anterooms 12% (90% PI 3–50%); designated tools per broiler house 7% (90% PI 1–18%). It is not possible to quantify the effects of combined control activities because the evidence‐derived estimates are inter‐dependent and there is a high level of uncertainty associated with each.info:eu-repo/semantics/publishedVersio

Evaluation of the application for new alternative biodiesel production process for rendered fat including Category 1 animal by-products (BDI-RepCat® process, AT)

A new alternative method for the production of biodiesel from rendered fat, including animal by‐product (ABP) Category 1 tallow, was evaluated. The method consists of a conversion phase, based on esterification and transesterification in a single step (at temperature ≥ 200°C, pressure ≥ 70 bar with a retention time ≥ 15 min), using MgO as a catalyst and in the presence of methanol (10–15%), followed by vacuum distillation (at ≥ 150°C, ≤ 10 mbar) of the end‐product, biodiesel and the co‐product, glycerine. Prions (PrPSc), which are abnormal isoforms of the prion protein, were considered by the applicant to be the most resistant hazard. In accordance with previous EFSA Opinions and current expert evaluation, a reduction in prion infectivity, or detectable PrPSc, of at least 6 log10 should be achieved for the process to be considered equivalent to the processing method laid down in the Regulation (EU) No 142/2011. Published data from an experimental replication of the conversion step of the biodiesel production process under consideration were provided, which showed an at least 6 log10 reduction in detectable PrPSc, by Western blot, in tallow that had been spiked with murine and human prion strains. In addition, it was demonstrated that the presence of methanol does not affect the recovery or detection of PrPSc from a biodiesel substrate. Based on scientific literature, the vacuum distillation step has been shown to be capable of achieving an additional 3 log10 reduction in PrPSc. Therefore, the proposed alternative method is considered to be at least equivalent to the processing method laid down in the legislation for the production of biodiesel from raw materials including Category 1 ABP.info:eu-repo/semantics/publishedVersio

Potential BSE risk posed by the use of ruminant collagen and gelatine in feed for non‐ruminant farmed animals

EFSA was requested to estimate the cattle bovine spongiform encephalopathy (BSE) risk (C‐, L‐ and H‐BSE) posed by ruminant collagen and gelatine produced from raw material fit for human consumption, or from material classified as Category 3 animal by‐products (ABP), to be used in feed intended for non‐ruminant animals, including aquaculture animals. Three risk pathways (RP) were identified by which cattle could be exposed to ruminant feed cross‐contaminated with ruminant collagen or gelatine: 1) recycled former foodstuffs produced in accordance with Regulation (EC) No 853/2004 (RP1), 2) technological or nutritional additives or 3) compound feed, produced either in accordance with Regulation (EC) No 853/2004 (RP2a) or Regulation (EU) No 142/2011 (RP2b). A probabilistic model was developed to estimate the BSE infectivity load measured in cattle oral ID50 (CoID50)/kg, in the gelatine produced from the bones and hide of one infected animal older than 30 months with clinical BSE (worst‐case scenario). The amount of BSE infectivity (50th percentile estimate) in a member state (MS) with negligible risk status was 7.6 × 10–2 CoID50/kg, and 3.1 × 10–4 CoID50/kg in a MS with controlled risk status. The assessment considered the potential contamination pathways and the model results (including uncertainties) regarding the current epidemiological situation in the EU and current statutory controls. Given the estimated amount of BSE infectivity to which cattle would be exposed in a single year, and even if all the estimated undetected BSE cases in the EU were used for the production of collagen or gelatine (either using raw materials fit for human consumption or Category 3 ABP raw materials), it was concluded that the probability that no new case of BSE in the cattle population would be generated through any of the three RP is 99–100% (almost certain).info:eu-repo/semantics/publishedVersio